FcRn and macropinocytosis promote albumin uptake in tumor-associated macrophages

Background: Serum albumin, the most abundant and long-circulating protein in serum, has been successfully used as a drug delivery vehicle for cancer therapy. We have previously demonstrated that albumin accumulates at high levels in tumors, including anaplastic thyroid cancer (ATC), pancreatic cancer and prostate cancer. Oncogene-driven macropinocytosis in cancer cells plays a major role in albumin uptake. However, little is known about the level of albumin uptake and the mechanisms that contribute to its accumulation in tumor-infiltrating immune cells. We hypothesized that tumor-infiltrating immune cells, particularly tumor-associated macrophages (TAMs), efficiently take up albumin via macropinocytosis and FcRn-mediated endocytosis. Methods: We assessed the in vivo uptake of mouse serum albumin by fluorescent reflectance imaging, and cellular albumin uptake by flow cytometry in the immunocompetent ATC TBP3743 ( Braf V600E p53 -/- ) in B6129SF1/J mice and melanoma YUMM1.7 ( Braf V600E/wt Pten -/- Cdkn2 -/- ) in C57BL/6J mice. We further identified tumor and cellular albumin uptake in neonatal Fc receptor (FcRn)- and caveolin 1 (Cav1)-deficient mice. In vitro cellular uptake of serum albumin was quantified in bone marrow-derived macrophages (BMDM) isolated from wildtype, FcRn -/- ( Fcgrt -/- ) and Cav1 -/- mice. We further treated BMDM with Na+/H+ exchanger (NHE) inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA) to block macropinocytosis, and fucoidan to block scavenger receptor-mediated endocytosis. Results: TBP3743 tumors and YUMM1.7 tumors accumulated serum albumin at 9%ID/g (% injected dose per gram tissue) and 11%ID/g, respectively, in mice 24 hrs after intravenous albumin administration. Although TAMs comprised only 4% of cells in ATC tumors and 5% in melanoma tumors, they accumulated a disproportionately high proportion (22% and 19%, respectively) of total fluorescent albumin in the tumor microenvironment. Tumors in Cav1 -/- and FcRn -/- mice accumulated only 10% and 29%, respectively, of the albumin uptake observed in wildtype mice. The mean fluorescence intensity of the serum albumin in TAMs in Cav1 -/- and FcRn -/- hosts was 78%and 32% of the level in wildtype hosts. In vitro Cav1 -/- and FcRn -/- BMDM accumulated albumin 107% and 44% of wildtype BMDM. EIPA reduced albumin uptake by 68%, 76% and 84% in wildtype, Cav1 -/- and FcRn -/- BMDM. Fucoidan didn't alter albumin uptake in either BMDM. Conclusion: TAMs exhibit the highest albumin uptake, on a per-cell basis, among cells within ATC and melanoma tumor microenvironments. Importantly, our data show that macropinocytosis and FcRn-mediated endocytosis are responsible for albumin uptake in TAMs. Thus, albumin accumulates efficiently in TAMs via FcRn and macropinocytosis, providing rationale for albumin as a drug delivery vehicle for TAM-targeted therapeutics.